The goal of this new R21 proposal is to develop a pro-inflammatory and non-tolerogenic HIV vaccine delivery system based on the dendritic cell targeting anti-DEC-205 antibody. The success of anti-DEC-205 as a stimulator of strong inflammatory immune responses depends on co-administration of non-specific dendritic cell maturation factors. In their absence, anti-DEC-205 induces antigen-specific tolerance rather than immunity. We hypothesize that regulatory T-cell epitopes contained in anti-DEC-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. This hypothesis is based on our discovery of a set of natural regulatory T-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory T cells. We have verified experimentally that these epitopes cause antigen-specific expansion of regulatory T cells and suppress inflammatory immune responses. We propose to develop a modified pro-inflammatory and non-tolerogenic anti-DEC-205 antibody. We expect that modification of regulatory T-cell epitopes will significantly diminish tolerogenicity, enabling use of anti-DEC-205 as a stand-alone HIV antigen delivery system that obviates the dangers associated with non-specific activation of the immune system. We will de-tolerize anti-DEC-205 by epitope modification using the process we developed to reduce immunogenicity of protein therapeutics. We will substitute key amino acids in the regulatory T-cell epitopes with those that are experimentally shown to interfere with MHC binding. We will then (1) produce a set of antibody variants recombinantly conjugated to HIV Gag, (2) identify de-tolerizing mutations that do not interfere with dendritic-cell targeting, and (3) and evaluate variants for reduced tolerogenicity, as well as for enhanced Gag immunogenicity. Finally, we will produce and characterize the immunogenicity of a de-tolerized anti-DEC-205-based HIV vaccine composed of immunogenic consensus sequences. PUBLIC HEALTH RELEVANCE: This project will develop an improved delivery vehicle for HIV vaccine components. Immune-dampening portions of the delivery vehicle will be silenced so that potent and effective immune responses can be raised against HIV.